Breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth

Abstract

Breast cancer immunotherapy is a potent treatment option, with antibody therapies such as trastuzumab increasing 2-year survival rates by 50%. However, active immunotherapy through vaccination has generally been clinically ineffective. One potential means of improving vaccine therapy is by delivering breast cancer antigens to dendritic cells (DCs) for enhanced antigen presentation. To accomplish this in vivo, we pseudotyped lentiviral vector (LV) vaccines with a modified Sindbis Virus glycoprotein so that they could deliver genes encoding the breast cancer antigen alpha-lactalbumin (Lalba) or erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) directly to resident DCs. We hypothesized that utilizing these DC-targeting lentiviral vectors asa breast cancer vaccine could lead to an improved immune response against self-antigens found in breast cancer tumors. Indeed, single injections of the vaccine vectors were able to amplify antigen-specific CD8T cells 4-6-fold over naïve mice, similar to the best published vaccine regimens. Immunization of these mice completely inhibited tumor growth in a foreign antigen environment (LV-ERBB2 in wildtype mice), and it reduced the rate of tumor growth in a self-antigen environment (LV-Lalba in wildtype or LV-ERBB2 in MMTV-huHER2 transgenic). These results show that a single injection with targeted lentiviral vectors can be an effective immunotherapy for breast cancer. Furthermore, they could be combined with other immunotherapeutic regimens to improve outcomes for patients with breast cancer.

Keywords: Alpha-lactalbumin; Breast cancer immunotherapy; Cancer vaccine; ERBB2/Her2; Lentiviral vector.

Fig. 1

ErbB2 and Lalba are potential antigens for lentiviral vaccine immunotherapy. (A) Meta-analysis of ERBB2 and LALBA mRNA expression in multiple tumor samples. Cancer Outlier Profile Analysis (COPA) [42] across 25 human breast cancer datasets identified ERBB2 and LALBA as highly overexpressed in a subset of tumor samples, with median gene ranks of 158 and 287, respectively. Brighter red indicates a more significant outlier score for a given dataset of tumor samples. Below, microarray mRNA expression levels from several representative datasets are shown [–45], demonstrating that ERBB2 or LALBA is overexpressed in a subset of patients with breast cancer. (B) Diagram showing the protein structure of the ERBB2 and LALBA genes. Signal peptides (SP) and transmembrane domains (TM) are indicated. (C) Diagram of the LV constructs used in the experiments (not to scale). The extracellular domain of ErbB2 and the entire coding sequence of mouse Lalba were cloned into a LV construct under the control of the ubiquitin promoter (Ubi). Oncomine™ (Compendia Bioscience™, part of Life Technologies™, Ann Arbor, MI) was used for analysis and visualization.

Fig. 2

Lentiviral vector vaccines generate a strong immune response to human ErbB2 in both foreign- and self-antigen environments. Intracellular cytokine staining was performed on splenocytes after stimulation with an ErbB2 peptide. The number of IFN-γ-expressing CD8+ cells as a percentage of total CD8+ cells was measured. (A) Representative flow cytometry plots show the percentage of IFN-γ⁺ cells after gating for CD8⁺CD4⁻ cells. The top panel shows Balb/cJ mice that were immunized with either the LV-ERBB2 or LV-GFP vaccine. The bottom panel shows MMTV-huHer2-Balb/cJ cross-bred mice immunized with the same conditions. (B) The ICCS data from all mice studied under the 4 conditions described. In both Balb/cJ and huHer2-Balb/cJ cross-bred mice, LV-ERBB2 immunization generated a higher antigen-specific T cell response than LV-GFP (t test, p < 0.001, p < 0.01 respectively).

Fig. 3

Lentiviral vector vaccines protect Balb/cJ mice against transplanted tumor challenge. Balb/cJ mice were challenged with 4T1.ERBB2 (A and B) or 4T1.Lalba (C and D) tumor cells, and tumor growth was measured over time. LV-ERBB2 immunization was performed prophylactically (A, 14 days before challenge) or therapeutically (B, 6 days after challenge). Tumor size in individual mice is plotted with solid light gray or red lines, while mean tumor size is plotted with dashed lines. A reduction in tumor growth was observed in prophylactic (p < 0.01) and therapeutic settings (p < 0.05, two-way ANOVA). (C and D) LV-Lalba immunization was performed prophylactically (14 days before challenge). Replicates and means are plotted in (C) as above. A survival curve is plotted in (D), using 1000 mm³ as the survival cutoff. A reduction in tumor growth was observed beginning at day 23 (p<0.01, t test), and median survival of immunized mice increased from 26 to 35 days (p<0.02, log-rank test).

Fig. 4

LV vaccines protect transgenic huHer2 mice against spontaneous tumor growth. (A) Transgenic MMTV-huHer2 mice were immunized with LV-ERBB2 or LV-GFP at 8 weeks of age. Tumor development in these mice was monitored for 80 weeks. (B) Survival is shown as the time to develop 750 mm³ tumors. LV-ERBB2 immunization led to long-term survival of > 80 weeks in the majority of mice, while LV-GFP led to early death in 75% of the mice (Gehan-Breslow-Wilcoxon, p < 0.05). (C) The rate of tumor growth was measured after first palpation, with all 3 observed tumors growing rapidly in the LV-GFP group, while 1 out of 2 tumors in the LV-ERBB2 immunized group developed much more slowly. (D) Human ERBB2 mRNA expression was measured from tumor extracts. Expression levels in the fast growing tumors (LV-GFP and LV-ERBB2 Fast) were higher than those in LV-ERBB2 Slow (t test, p < 0.02), while expression in LV-ERBB2 Slow was higher than background levels (vs. E0771, t test, p < 0.04).